1. Chapter 4: Part 2 Protein 3-D structure: 3 o and 4 o structure and protein folding.

2. 3 o Structure third level of protein organization folding of polypeptide chain causes 2 o structures to interact formation of motifs and domains

3. Proteins with similar 1 o structure also have similar 3 o structure tuna 1 GDVAKGKKTFVQKCAQCHTVENGGKHKVGPNLWGLFGRKTGQAEGYSYTDANKSKGIVWN yeast 1 GSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTDANIKKNVWDE rice 1 GNPKAGEKIFKTKCAQCHTVDKGAGHKQGPNLNGLFGRQSGTTPGYSYSTANKMAVIWEE tuna 61 ETLMEYLENPKKYIPGTKMIFAGIKKKGERQDLVAYLKSATS yeast 61 NNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACE rice 61 NTLYDYLLNPKKYIPGTKMVFPGLKKPQERADLISYLKEATS

4. Common Motifs

5. Motifs Combine to form Domains Hydrophobic interactions are the major driving force in folding domains Alpha/beta barrel Parallel twisted sheet Domains are independent folding units in a 3 o structure of a protein Individual domains have specific function

6. lactate dehydrogenasemalate dehydrogenase COO- C H 2 C O COO- COO- CHHO CH 2 COO- 3 COO- CH CH HO COO- C CH 3 O + NADH + NAD + + NADH + NAD + Protein family members share common domain structures

7. 4 o Structure Quaternary structure describes the organization of subunits in a protein with multiple subunits (oligomeric protein) Can have homo-multimers or hetero-multimers 2222  2 

8. Determine molecular weight of native protein by gel permeation chromatography Determine molecular weight of individual subunits by SDS-PAGE Can use the information to determine subunit composition 4 o Structure If……. Native protein – 160,000 daltons and  -Subunit – 50,000 daltons  -Subunit – 30,000 daltons Then…… Protein can have  2  2 structure

9. Subunits held together by non-covalent interactions Oligomeric protein is more stable than disassociated subunits Active site often made up of AA residues from different subunits 4 o and 3 o structure is often affected by ligand (substrate or inhibitor) binding. Important in enzyme regulation 4 o Structure

10. Tm Protein denaturation Denaturation – disruption of native conformation Heat commonly used to denature proteins Tm = temperature where 50% folded/50% unfolded. Typical Tm = 40-60 o C Tm for thermophiles >100 o C (Taq DNA polymerase) Chemical denaturants Chaotrophic agents = Urea, KCN detergents = SDS

11. Protein Folding Ribonuclease A (RNase A) will refold to native structure spontaneously (1 minute) >10 50 possible conformations If 10 -13 sec per conformation would take 10 30 years to sample enough to determine structure How do proteins fold so quickly?

12. Factors driving protein folding Conformational entropy A+B  C decreases entropy (unfavorable) Non-covalent interactions give favorable enthalpy value Hydrophobic effect increases entropy by freeing water (favorable)  G =  H - T  S - +

13. Protein Folding Structures of globular proteins are not static Proteins “breathing” between different conformations Proteins fold towards lowest energy conformation Multiple paths to lowest energy form All folding paths funnel towards lowest energy form Local low energy minimum can slow progress towards lowest energy form

14. Pathway of Protein Folding 1) Nucleation of folding - Rapid and reversible formation of local 2 o structures form 2) Formation of domains (Molten Globular intermediates) through aggregation of local 2 o structures 3) Domain conformations adjust to form native protein

15. Chaperonins Protein complexes that promote protein folding Chaperonins don’t determine native structure Prevent misfolding and aggregation of protein Sequesters unfolded protein from other proteins Require ATP for protein binding, after ATP hydrolysis native protein released Thought to bind unfolded regions of protein

16. Disulfides Bonds Stabilize native structure Formed after native conformation achieved Abundant in secreted proteins but not in intracellular proteins Protein disulfide isomerase catalyzes reduction of incorrect disulfide linkages